Purification of pertussis antigens

ABSTRACT

A process is provided for the production of lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA) and at least one fimbrial agglutinogen from a liquid culture of Bordetella pertussis, which comprises the steps of (a) separating the culture into cellular and supernatant fractions, (b) concentrating the supernatant fraction, (c) fractionating the concentrated supernatant fraction to isolate LPF and FHA containing fractions, and (d) isolating at least one fimbrial agglutinogen from the cellular fraction. A vaccine composition may be produced by mixing so-produced LPF, FHA and fimbrial agglutinogens produced.

This invention relates to a process for the production of antigenicsubstances for use in formulating vaccines against infection byBordetella pertussis.

Hitherto, widespread use has been made of killed whole-cell Bordetellapertussis vaccines in the control of pertussis. Adverse reactions towhole-cell vaccines and the resulting reduced public acceptance of thesevaccines has led to considerable research being carried out in anattempt to produce a safer acellular vaccine containing isolatedpertussis antigens.

Three particular antigen fractions are regarded as being usefulcomponents of acellular vaccines, namely (1) lymphocytosis promotingfactor (LPF), (2) filamentous haemagglutinin (FHA) and (3) fimbrialagglutinogens (fimbriae).

Included in the class of antigens represented by fraction (3), i.e. thefimbrial agglutinogens, are the agglutinogens referred to as"agglutinogens 2 and 3". (Agglutinogens 2 and 3 may also be referred toas "Ag₂ +3"). Agglutinogens 2 and 3 may be regarded as consisting offimbriae isolated from organisms bearing at least agglutinogen 2 and 3antigens. (Agglutinogen 3 is referred to as "agglutinogen 6" by certainworkers and it may be that agglutinogens 3 and 6 are the same).

Although procedures are known for isolating these fractions on a smallscale, no procedures have hitherto been available for producing allthree fractions separately and efficiently from cultures of Bordetellapertussis. Thus, for example, EP-A-No.0003916 describes a process forobtaining lymphocytosis promoting factor (LPF) by subjecting a liquidpreparation derived from cells of Bordetella pertussis to affinitychromatography utilizing as a stationary phase a sialo-protein. Howeverthe liquid preparation which was used was derived from a homogenizedcell paste and while the procedures described are efficient for theproduction of LPF, the patent is not directed to the separate isolationof filamentous haemagglutinin (FHA) and agglutinogens 2 and 3 (Ag₂₊₃).

Procedures have also been proposed for obtaining lymphocytosis promotingfactor (LPF) and filamentous haemagglutinin (FHA) from the culturesupernatant of Bordetella pertussis, for example Sato et al (Infectionand Immunity, July 1983, Vol. 41, p. 313-320) describe the purificationof LPF and FHA from culture supernatants by differential absorption on ahydroxyl apatite column.

These prior documents are not concerned, however, with the co-productionof all three of lymphocytosis promoting factor (LPF), filamentoushaemagglutinin (FHA) and fimbrial agglutinogens.

For the production of vaccine compositions comprising all three of theseantigen fractions it would be desirable to obtain the three desiredfractions in comparable yields since it may be advantageous for thefractions to be present in approximately equal proportions in theeventual vaccine.

According to the present invention there is provided a process for theproduction of lymphocytosis promoting factor (LPF), filamentoushaemagglutinin (FHA) and at least one fimbrial agglutinogen from aliquid culture of Bordetella pertussis, which comprises the steps of

(a) separating the culture into cellular and supernatant fractions,

(b) concentrating the supernatant fraction,

(c) fractionating the concentrated supernatant fraction to isolate LPFand FHA containing fractions, and

(d) isolating at least one fimbrial agglutinogen from the cellularfraction.

Preferably in step (d), fimbrial agglutinogens comprising at leastagglutinogens 2 and 3 (Ag₂₊₃) are isolated from the cellular fraction.Optionally, the isolated agglutinogens additionally include one or moreof agglutinogens 4, 5 and 6.

The invention also provides a process for the production of a vaccinecomposition comprising lymphocytosis promoting factor (LPF), filamentoushaemagglutinin (FHA) and at least one fimbrial agglutinogen whichcomprises mixing (i) LPF, (ii) FHA and (iii) at least one fimbrialagglutinogen, said LPF, FHA and at least one fimbrial agglutinogen beingproduced by steps (a) to (d) described above and being detoxified priorto or subsequent to mixing. Preferably the fimbrial agglutinogensinclude at least agglutinogens 2 and 3 (Ag₂₊₃). The fimbrialagglutinogens may also include at least one of agglutinogens 4, 5 and 6.

By isolating the LPF, FHA and the at least one fimbrial agglutinogen inaccordance with the invention it is possible to obtain these componentsfrom a single liquid culture in yields enabling their recombination intherapeutically effective quantities in vaccine compositions.

As indicated, in carrying out the process of the invention thesupernatant fraction is concentrated prior to carrying out fractionationstep (c) and surprisingly it has been found that by carrying thissequence of steps, i.e. with the concentration step (b) carried outafter separation of the supernatant fraction from the cellular fraction,unacceptable losses of FHA can be avoided.

The separation of the culture into cellular and supernatant fractions ispreferably carried out by centrifugation when the pH of the culture isgreater than 7.0 and preferably at a pH greater than or equal to 8.0.Most preferably the pH of the culture is in the range of 7.5 to 9.0.

After centrifugation, the supernatant fraction is preferablyconcentrated so as to reduce its volume to less than 50% and preferablyless than 25% of the original volume. Conveniently the concentration maybe effected by any conventional dewatering means, for example byPellicon concentration. Following the concentration step and prior tofractionating the supernatant into LPF and FHA containing fractions thesupernatant is preferably subjected to membrane filtration to remove anyresidual organisms. Fractionation step (c) can then conveniently becarried out by contacting the supernatant with hydroxyl apatite, forexample in a column, whereupon an FHA containing fraction is retainedand an eluate comprising an LPF containing fraction can be recovered.

The LPF containing fraction may be purified by conventional proteinfractionation techniques, for example by precipitating proteinaceousmaterial by increasing the salt concentration using for example ammoniumsulphate followed by extraction of the precipitate using a suitablebuffer. The extract may then be dialysed and the dialysed LPF-containingsolution so obtained may be further purified to remove other proteinsand lipopolysacchoride, for example by the procedure described inEP-A-No. 0003916.

Thus, for example, the dialysed LPF-containing solution may be appliedto a fetuin sepharose column and retained LPF eluted using a magnesiumchloride buffer.

The thus purified LPF containing fraction may then be dialysed again,filtered and then subjected to a detoxifying procedure.

Purification of the FHA containing fraction may be achieved by elutingthe fraction from the hydroxyl apatite adsorbent using buffers ofincreasing ionic strength followed by conventional protein purificationsteps including, for example, precipitation at high salt concentrationsand chromatography. Finally, the purified FHA-containing fractions maybe subjected to membrane filtration and then to a detoxifying step.

In order to isolate fimbrial agglutinogens, e.g. agglutinogens 2 and 3(Ag₂₊₃) from the cellular fraction, the cells are preferably washed andthen homogenized in a suitable buffer. Following centrifugation, thesupernatant may then be subjected to conventional protein purificationprocedures in order to isolate a fraction comprising fimbrialagglutinogens. Thus, for example, a fimbrial agglutinogen-containingfraction may be precipitated by increasing the ionic strength of thesolution, followed by one or more extractions with buffer,reprecipitations and dialysis. Finally, the purified fimbrialagglutinogen-containing fraction may be subjected to membrane filtrationfollowed by a detoxifying step.

If desired the lipopolysaccharide content of the fimbrial agglutinogenfraction may be reduced by affinity chromatography, e.g. on apolymyxin-sepharose 4B column.

The detoxifying steps are preferably carried out by treating the LPF,FHA and fimbrial agglutinogen containing fractions individually or incombination with a conventional toxoiding agents such as, for exampleformaldehyde.

In order to produce vaccine compositions from the LPF, FHA and fimbrialagglutinogen fractions, these fractions are combined in therapeuticallyeffective proportions and formulated into dosage units containing forexample at least 1-5 and preferably at least 2 ug of each component perunit dose.

Conventional growth media may be employed to produce the liquid cultureof Bordetella pertussis used as starting material in the process of theinvention. However in order to produce commercially valuable yields ofFHA it has been found to be desirable to use Stainer and Scholte'smedium containing 2-6 mg/ml of dimethyl-B-cyclodextrin (MeCD),particularly in shaken cultures or stirred fermenters. Growth of thebulk culture may conveniently be carried out in 1 litre Thompson bottlescontaining 300 ml of medium or in 10 litre fermenters. A suitable strainof Bordetella pertussis is the Wellcome 28 strain which is of the 1.2.3serotype. Other strains of a 1.2.3 serotype which produce adequateyields of antigens may be used.

The production of lymphocytosis promoting factor (LPF), filamentoushaemagglutinin (FHA) and agglutinogens 2 and 3 (Ag₂₊₃) in accordancewith the invention will now be described by way of example.

EXAMPLE

A culture of Bordetella pertussis was prepared as follows:

A freeze-dried ampoule of B. pertussis, strain Wellcome 28, was opened,the contents dispersed in sterile water and pipetted on to a charcoalagar plate containing 10% defibrinated horse blood. After incubation at35° C. for 48 hours the organisms were subcultured on to severalcharcoal agar plates which were incubated at 35° C. for 48 hours.

The organisms were then scraped into several 250 ml conical flaskscontaining 100 ml Stainer and Scholte's medium supplemented with 1%casamino acids and 1 mg MeCD/ml. The flasks were shaken orbitally (180rev./min) for 24 hours at 35° C. and then 5-10 ml transferred to each of60 Thompson bottles containing 300 ml medium. The bottles were shaken ona gently reciprocating shaker at 35° C. for 48 hours before pooling thecontents of the bottles and harvesting.

The resulting liquid culture of Bordetella pertussis was centrifuged ina Sorvall RC3B centrifuge for one hour at 5000 rpm. The supernatant wasdecanted and the cells stored at 4° C. for preparation of agglutinogens2 and 3 (Ag₂₊₃).

The supernatant was concentrated using a Millipore Pellicon apparatusfitted with a 10,000 molecular weight cut-off filter. Eighteen liters ofsupernatant was concentrated to a volume of approximately 4 liters. Theconcentrate was then subjected to sterile filtration through a Gelman0.2 um polysulphone mini capsule filter. 1. Purification of FilamentousHaemagglutinin (FHA)

Purification of the filamentous haemagglutinin (FHA) from the culturesupernatant was achieved by consecutive steps of hydroxy apatitechromatography, ammonium sulphate precipitation and Sepharose CL-6Bchromatography.

A. Hydroxylapatite Chromatography

400 g of spheroidal hydroxyapatite (BDH Chemicals Ltd) were suspended in500 ml of 0.1M NaOH. The powder was allowed to settle for about 20minutes at room temperature and excess liquid decantaed. The washingwith sodium hydroxide was repeated twice, followed by repeated washingwith distilled water until the pH of the eluate was about 8.0.

The washed powder was suspended in 500 mls of 0.01M phosphate buffer andafter 20 minutes the buffer was decanted. The washing with phosphatebuffer was repeated three times.

The washed hydroxylapatite was packed into a chromatography columnequilibrated with 0.01M phosphate buffer at pH 8.0

The column was then connected to a reservoir containing the concentratedsupernatant which was pumped through the column using a peristaltic pumpat a flow rate of about 500 ml/hour. The eluate was retained forisolation of LPF (see below).

The retained FHA fraction was eluted from the column by washingsuccessively with (i) 0.01M phosphate buffer at pH 8, (ii) 0.1Mphosphate buffer at pH 8 and (iii) 0.1M phosphate-0.5M sodium chloridebuffer at pH 6.5. During the final washing seventy 8 ml fractions werecollected and the optical density at 280 mm of each fraction wasrecorded. Also, the haemagglutinating activity of alternate fractionswas assayed using freshly washed goose erythrocytes. Fractions having ahaemagglutinating titre (Log 2) greater than or equal to 7 were pooled.A record of the elution is shown in FIG. 1.

B. Ammonium Sulphate Precipitation

FHA was precipitated from the pooled fractions by adding ammoniumsulphate to give a 30% saturated solution, followed by centrifugation.The supernatant was decanted and discarded and the precipitate wasdissolved in 0.05M phosphate-0.5M sodium chloride buffer at pH 7.2 anddialysed against the same buffer. The dialysed suspension wascentrifuged and the supernatant retained for subsequent purification.

C. Sepharose CL-6B Chromatography

The supernatant was then subjected to chromatography on Sepharose CL-6Bgel which had previously been equilibrated with 0.05M phosphate-0.5Msodium chloride buffer at pH 7.2

The FHA was then eluted from the column using 0.05M phosphate-0.5Msodium chloride buffer at pH 7.2 and eighty 5 ml fractions werecollected. The eluate fractions were monitored for protein andhaemagglutinating activity in the manner described above and fractionshaving an haemagglutinating titre greater than or equal to 7 werepooled. A record of the elution is shown in FIG. 2. 2. Purification ofLPF

The eluate from the hydroxyapatite chromatography step was then treatedfor recovery of LPF.

A. Ammonium Sulphate Precipitation

In an initial step, impure LPF was precipitated by adding ammoniumsulphate to give 74% saturated solution. The resulting suspension wascentrifuged and the supernatant discarded. The precipitate wasresuspended in 0.05M phosphate-0.05M sodium chloride buffer at pH 7.2.The suspension was then centrifuged at 15,000 rpm and the supernatantretained. The product was extracted a further four times using the samebuffer and the resulting supernatants pooled.

B. Fetuin Sepharose Chromatography

The pooled supernatants were then dialysed against 0.05M phosphate-0.05Msodium chloride buffer at pH 7.2 and then subjected to chromatography ona fetuin sepharose gel which had been pre-equilibrated in 0.05Mphosphate-0.05M sodium chloride buffer at pH 7.2. The column was thenwashed with the same buffer and the eluate discarded.

A purified LPF-containing fraction was then eluted from the column using6.7 mM tris-0.013M sodium chloride/3M magnesium chloride buffer at pH6.4 and thirty 50-drop fractions were collected. Fractions having anhaemagglutinating titre greater than or equal to 7 were pooled anddialysed against 2 liters of 0.05Mtris HC1, IM NaCl buffer at pH 8.0.The LPF containing fraction was then dialysed again using 0.05Mphosphate-0.5M sodium chloride buffer of pH 7.2 and the resultingpurified LPF-containing fraction retained. 3. Preparation ofAgglutinogens 2 and 3 (Ag₂ +3)

The centrifuged cellular fraction was washed using sterile pyrogen-freedistilled water, centrifuged and then homogenized using 0.014Mphosphate-0.14M sodium chloride buffer at pH 7.2. The homogenizedbacterial suspension was then centrifuged at 9000 rpm to removebacterial cells and the supernatant which contained Bordetella pertussisfimbrae retained. Ammonium sulphate was added to the supernatant to givea final concentration of 30% saturation and the suspension stored at 4°C. overnight to precipitate fimbrial proteins. After centrifuging at9000 rpm the supernatant was discarded and the pellet extracted usingpre-cooled phosphate buffer. The suspension was centrifuged at 15,000rpm and the supernatant retained. The extraction was repeated four timesand the resulting supernatants pooled.

Ammonium sulphate was added to the pooled supernatants to give a finalconcentration of 15% saturation and the resulting suspension storedovernight at 4° C. to precipitate fimbrial proteins.

The suspension was centrifuged at 15,000 rpm and the supernatantrejected.

The pellet was extracted with phosphate buffer and centrifuged at 15,000rpm. The supernatant was collected and the extraction repeated fourtimes and the resulting supernatants pooled.

Ammonium sulphate was added to the pooled supernatants to give a finalconcentration of 15% saturation and the resulting suspension storedovernight at 4° C. to precipitate fimbrial proteins. The suspension wascentrifuged at 15,000 rpm and the supernatant rejected. (Furtherammonium sulphate precipitations may be employed if necessary).

The pellet was extracted with phosphate buffer and centrifuged at 15,000rpm. The supernatant was collected and the extraction repeated fourtimes and the resulting supernatants pooled.

The pooled supernatant was then dialysed against phosphate buffer andsubjected to membrane filtration.

4. Detoxification

All antigens were filtered through a Millex GV 0.22 um filter unit. TheLPF preparation was diluted to a final protein concentration of 200ug/ml using 0.05M phosphate-0.5M sodium chloride buffer at pH 7.2. A 40%formaldehyde solution was added to give a final concentration offormaldehyde of 0.5%. The LPF-containing fraction was then incubated at37° C. for 14 days, inverting the contents at least once every 2 days todisperse any precipitate formed.

The resulting detoxified LPF fraction was then dialysed against PBSbuffer containing 0.01% formaldehyde and 0.01% thiomersal and decantedinto a container for storage, ensuring that all precipitated materialhas been transferred.

A similar detoxification procedure was applied to the FHA fraction andthe agglutinogens 2 and 3 (Ag₂₊₃) fraction, except that the incubationat 37° C. was for 7 days.

The detoxified antigens in PBS containing 0.01% formaldehyde 0.01%thiomersal were mixed in equal proportions and diluted with sterilewater, 4 x concentrated PBS containing 0.04% formaldehyde and 0.04%thiomersal and alhydogel. The resultant vaccine, in isotonic PBS,contains 120 ug protein/ml, 25% Alhydrogel, 0.01% formaldehyde and 0.01%thiomersal. As an alternative adjuvant to Alhydrogel, aluminiumphosphate (added as aluminium chloride) may be employed. The vaccine canbe further diluted with PBS, diptheria and tetanus toxoids andalhydrogel, to yield a concentration of pertussis antigens to 60 ug/ml.

The following yields of FHA, LPF and agglutinogens 2 and 3 (AG₂₊₃) wereobtained from 18 liters of culture:

FHA--190 mg

LPF--50 mg

agglutinogens 2 and 3 (Ag₂₊₃)--50 mg

We claim:
 1. A process for the co-production of lymphocytosis promotingfactor (LPF), filamentous haemagglutinin (FHA) and at least one fimbrialagglutinogen from a liquid culture of Bordetella pertussis, whichcomprises the steps of(a) separating the culture into cellular andsupernatant fractions, (b) subsequent to step (a) concentrating thesupernatant fraction to less than 50% of its original volume to form aconcentrated aqueous supernatant fraction of reduced water content, (c)subsequent to step (b), fractionating the concentrated supernatantfraction to isolate LPF and FHA containing fractions, and (d) isolatingat least one fimbrial agglutinogen from the cellular fraction.
 2. Aprocess according to claim 1 wherein the fimbrial agglutinogens isolatedin step (d) include at least one of agglutinogens 2, 3, 4, 5 and
 6. 3. Aprocess according to claim 1 wherein the fimbrial agglutinogens isolatedin step (d) comprise at least agglutinogens 2 and 3 (Ag₂₊₃).
 4. Aprocess according to claim 3 wherein the isolated agglutinogensadditionally include one or more of agglutinogens 4, 5 and
 6. 5. Aprocess according to claim 1 wherein separation step (a) is carried outat a pH greater than 7.0.
 6. A process according to claim 5 whereinseparation step (a) is carried out at a pH in the range from 7.5 to 9.0.7. A process according to claim 1 wherein in step (b) the supernatant isconcentrated to less than 50% and preferably less than 25% of itsoriginal volume.
 8. A process according to claim 1 wherein in step (c)the LPF containing fraction is purified by adsorption on fetuinsepharose followed by elution using a magnesium chloride buffer.
 9. Aprocess for the production of a vaccine composition comprisinglymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA)and at least one fimbrial agglutinogen which comprises mixing (i) LPF,(ii) FHA and (iii) at least one fimbrial agglutinogens producedaccording to claim 1, the LPF, FHA and the at least one fimbrialagglutinogens being detoxified prior to or subsequent to mixing.